Dominique Laurain-Mattar

LCMS and GCMS for the screening of alkaloids in natural and in vitro extracts of Leucojum aestivum.

HPLC coupled to a mass spectrometer (MS) was used for the analysis of galanthamine and lycorine in natural extracts of Leucojum aestivum and in their in vitro cultures grown with a precursor (ACC), inhibitors (AgNO(3), STS), or an absorber (KMnO(4)) of ethylene. The maximum galanthamine (0.002%) and lycorine (0.02%) concentrations in tissue cultures were obtained in the presence of KMnO(4). GCMS was used to investigate underivatized alkaloid mixtures from L. aestivum. Seven alkaloids were identified in in vivo bulbs. KMnO(4) led to the highest diversity of alkaloids in tissue culture extracts.

Hairy root and tissue cultures of Leucojum aestivum L.—relationships to galanthamine content

Galanthamine, an isoquinoline alkaloid acetylcholinesterase inhibitor, is an important agent used all around the world for the symptomatic treatment of senile dementia of the Alzheimer’s type. The production of this metabolite and the availability of the plant are limited and prompted the search for an alternative way to obtain this valuable metabolite using in vitro cultures of Leucojum aestivum L. It is known that cell differentiation level shows a major influence upon the accumulation of alkaloids. For this reason, tissue cultures of L. aestivum showing different stages of morphogenesis controlled by exogenous growth regulators were established. Agrobacterium rhizogenes strain LBA 9402 has been tested for its capacity to induce hairy roots of this monocotyledonae plant.

Leucojum aestivum plants propagated in in vitro bioreactor culture and on solid media containing cytokinins

Cytokinins are growth regulators that stimulate cell division and control morphogenesis in plants, however their role in regulating secondary metabolism is not well studied. The influence of various cytokinins (benzyladenine, zeatin, kinetin, meta‐topolin, thidiazuron) and culture systems (solid and temporary immersion RITA® system) on the quality Leucojum aestivum plant regenerated from somatic embryos was investigated. The largest number of regenerated plants (181.6 and 168.8) was obtained from the embryos cultivated on media enriched with meta‐topolin and benzyladenine. Thidiazuron and meta‐topolin led to the highest number of normally developed plants (94.8 and 90.6). The random amplified polymorphic DNA analysis of in vitro and in vivo plants showed four clusters of similarity. The highest biomass (growth index: 2.49) was obtained with the temporary immersion RITA® system. Alkaloid extracts were analyzed by LC‐MS, leading to the quantification of galanthamine and lycorine both in plant materials and in liquid media. The highest contents of galanthamine (0.05% dry weight) were observed in plants cultivated in the presence of thidiazuron in bioreactor system. Galanthamine was accumulated (highest content 0.05% dry weight) in plants cultivated in the presence of thidiazuron in bioreactor system whereas lycorine was synthetized mainly in plants cultivated on solid media.

Influence of auxins on somatic embryogenesis and alkaloid accumulation in Leucojum aestivum callus

In vitro cultures of Leucojum aestivum are considered as an alternative for the production of galanthamine, which is used for the symptomatic treatment of Alzheimer’s disease. We studied the effects of auxins 2,4-dichlorophenoxyacetic acid (2,4-D), 4-amino-3,5,6-trichloropicolinic acid (picloram), 3,6-dichloro-o-anisic acid (dicamba) at concentrations of 25 and 50 µM on the induction of embryogenic callus and its capacity to induce somatic embryogenesis and alkaloid accumulation. The embryogenic response of the explants was from 30% for 25 µM of dicamba to 100% for picloram (for both 25 and 50 µM). 2,4-D (50 µM) stimulated greater callus proliferation and somatic embryo induction as compared to the other auxins. Polyethylene glycol (PEG) stimulated somatic embryo maturation. Callus grown on media containing 50 µM of auxins produced fewer phenolic compounds as compared with callus grown on media containing 25 µM of auxins. GC-MS analyses showed seven alkaloids in the in vivo bulbs and two to four in callus culture. Galanthamine was detected in callus cultivated with 2,4-D (25, 50 µM), picloram (25 µM), and dicamba (50 µM). Other alkaloids, trisphaeridine, tazettine, and 11-hydroxyvittatine were accumulated only in callus growing on medium with picloram (50 µM).

Production of Alkaloids in Plant Cell and Tissue Cultures

A low or no productivity of alkaloids in plant cell cultures can be explained by an insufficient level of cell differentiation. The first strategy described in this chapter for improving isoquinoline alkaloid accumulation is organogenesis and somatic embryogenesis induced by the addition of exogenous growth regulators in Papaver somniferum and Leucojum aestivum cell cultures. The second strategy described is the transformation of medicinal plants (Atropa belladonna, Papaver somniferum, and Leucojum aestivum) using Agrobacterium rhizogenes to form hairy root cultures, which carry with them with the benefits of fast growth and rates of alkaloid production equal to or greater than that found for the intact plant.

4′‐O‐Methylnorbelladine feeding enhances galanthamine and lycorine production by Leucojum aestivum L. shoot cultures

Galanthamine and lycorine are Amaryllidaceae alkaloids that exhibit a wide range of pharmacological activities. Organic synthesis and plant extraction are the two main commercial sources of galanthamine. Since total organic synthesis is low yielding and complex, and the harvesting of plant biomass is causing wild plant depletion, in vitro cultures offer an alternative approach. Amaryllidaceae alkaloids can be regarded as derivatives of the common precursor 4′‐O‐methylnorbelladine via intramolecular oxidative phenol coupling. The aim of this work was to enhance the biosynthetic pathway of these alkaloids using in vitro techniques. The precursor 4′‐O‐methylnorbelladine was incorporated into the liquid medium of Leucojum aestivum shoot cultures at different concentrations (0, 0.05, 0.1, or 0.2 g/L). The cultures were conducted for various periods of time (15, 30, and 40 days). After alkaloid extraction and according to LC‐ESI‐MS analysis, the precursor highly stimulated the biosynthesis of both galanthamine (0.5 mg/g dry weight [DW], V/S 0.01 mg/g DW in the control sample, i.e. without precursor) and lycorine (0.2 mg/g DW, V/S 0.04 mg/g DW in the control sample) after its biotransformation by the shoot cultures. The optimal culture conditions for the production of both alkaloids were 0.1 g/L of precursor and 15 days of culture.

Toxicity of Hydnora johannis Becca. dried roots and ethanol extract in rats

AIM OF THE STUDY Hydnora johannis Becca. (Hydnoraceae) commonly is used for the treatment of dysentery, diarrhoea, cholera and swelling tonsillitis in the folk medicine of Sudan and other African countries. This study evaluates the toxicological effects of Hydnora johannis roots on Wistar rats. MATERIALS AND METHODS Rats were randomized into control, groups fed with 2, 10, 20% of dried roots for 8 weeks and other groups given ethanol extract (50, 100, 200 and 400mg/kg/day) through oral and intramuscularly administration for 2 weeks. Toxicity was evaluated using biochemical and histopathological assays. RESULTS Alterations in the levels of aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, cholesterol and urea were observed. Histopathological analysis revealed that the toxic effect were mainly on the liver, kidney and spleen on all treated groups. However, the impact of the dried roots was mild compared to the ethanol extract. Remarkably, there was a drop in cholesterol level in all treatment groups suggesting the antiartherogenic effect of Hydnora johannis roots. CONCLUSION The results from this study suggest that the powder preparation as well as ethanolic extract of Hydnora johannis roots induced toxic effect on Wistar rats. The observed toxic effect might be due to the dose and/or frequency of administration. Although in traditional medicine the extract is administrated in low dose, the results suggest the necessity of standardization of the drug.

Stimulating effect of both 4’-O-methylnorbelladine feeding and temporary immersion conditions on galanthamine and lycorine production by Leucojum aestivum L. bulblets

Bulb cultures of Leucojum aestivum and L. aestivum ‘Gravety Giant’ were subcultured in medium containing the precursor 4’‐O‐methylnorbelladine (MN) at various concentrations [0 (control), 0.15 and 0.3 g/L]. The cultures were conducted in bioreactor RITA® and lasted for 15, 30, 40 and 50 days. The growth rate and the alkaloid accumulation in bulblets were studied. For this latter purpose, a purification method was developed. It comprised a highly selective solid phase extraction using on the one hand, UPTI‐CLEAN SI and SCX cartridges for plant extracts and on the other hand, 2H cartridges for culture media. Pure alkaloidal fractions were, thus, analyzed by LC‐ESI‐MS allowing the quantitative evaluation of galanthamine and lycorine from culture extracts. Precursor feeding along with temporary immersion conditions was found to significantly improve the accumulation of both galanthamine and lycorine. The maximal concentrations of galanthamine (0.81 mg/g DW) and lycorine (0.54 mg/g DW) in L. aestivum bulblets were reached, respectively, after 40 days of culture with 0.15 g/L of precursor and after 30 days of culture with 0.3 g/L of precursor. In L. aestivum ‘Gravety Giant’ bulb cultures, 0.3 g/L of precursor was the best condition for both galanthamine (0.6 mg/g DW after 50 days) and lycorine (1.13 mg/g DW after 30 days).